A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2Adel30, was found previously to be generally well-tolerated in humans, but a rash and an elevation of liver enzymes in serum occurred in some vaccinees. 2Adel30, a non-temperature-sensitive virus, contains a 30 nucleotide deletion (del )in the 3' untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 has been utilized to generate attenuating mutations which may be useful to further attenuate the 2Adel30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and from a panel of 1,248 clones that were isolated in Vero cells, twenty temperature-sensitive (ts) mutant viruses were identified which were ts in both simian Vero and human liver HuH-7 cells (n = 13) or in only HuH-7 cells (n = 7). Each of the twenty ts mutant viruses possessed an attenuation (att) phenotype as indicated by restricted replication in the brains of seven-day-old mice. Since the 2Adel30 vaccine candidate retains a low level of virulence for the liver and since other findings support the ability of dengue viruses to infect hepatocytes and cause liver pathology, we sought to develop mutations that would selectively restrict replication of dengue 4 virus in liver cells. Toward this end, we identified seven mutant viruses which have a HuH-7 cell-specific ts phenotype. The mutations present in these viruses are the first reported for DEN viruses that confer restricted replication in liver cells and may be helpful in limiting virus replication and pathology in the liver of vaccine recipients. The contribution of single mutations identified in the HuH-7 cell-specific ts viruses to the observed phenotypes is presently being assessed by introduction of the individual mutations into recombinant DEN4 viruses. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and non-structural genes as well as in the 5' and 3' UTR with more than one change occurring, in general, per mutant virus. The determination of the complete genomic sequence of 20 DEN4 viruses in the present study provided an opportunity to identify mutations in the NS genes and the UTRs resulting from passage in Vero cells. Such mutations are well-suited for inclusion in DEN4 antigenic chimeric viruses bearing the structural proteins of DEN1,-2, or 3. It was also found that identical mutations were found in multiple sister 5-FU mutant viruses, each of which had several passages in Vero cells, and these common mutations may represent adaptive changes that confer an increased efficiency of DEN4 replication in Vero cells. Such mutations would be potentially beneficial for inclusion in a live-attenuated DEN virus vaccine by increasing the yield of vaccine virus during manufacture. Interestingly, three distinct amino acid substitutions were found in NS4B of several of these 5-FU viruses bearing the common "Vero cell adaptation" mutations. The exact function of this gene is unknown. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the del30 deletion mutation, thereby creating rDEN4del30-4995, a recombinant virus which is ts and more attenuated than rDEN4del30 in the brains of mice. A menu of attenuating mutations is being assembled that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.